ES2581591A1 - Procedure for obtaining a natural extract of acorn peel, extract obtained and its use (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Procedure for obtaining an extract of acorn peel, extract obtained and its use. The present invention relates to a process for obtaining an acorn peel extract comprising a) shredding the shell, b) adding to the shredding of the shell obtained in step a) a solvent selected from the group consisting of water, alcohol c1 -c6 and mixtures thereof, c) stirring the mixture of the triturate and the solvent to obtain two phases, and d) separating the two phases obtained in step c) and collecting the supernatant comprising the extract. Likewise, it refers to the acorn peel extract obtainable by said procedure and its use as an antioxidant. (Machine-translation by Google Translate, not legally binding)

Description

Procedure for obtaining a natural acorn shell extract, extract obtained and its use.

Object of the invention 5

The present invention belongs to the field of obtaining natural antioxidants. Specifically, it refers to a procedure for obtaining an acorn shell extract, the extract obtained by that procedure and its use, in particular as an antioxidant.

Background of the invention

Protection against oxidative deterioration of various types of food is a clear concern in the food industry. Antioxidants can act in various ways during oxidation (for example by sequestering metal ions, capturing free radicals, acting as reducing agents, decomposing peroxides, etc.). In food systems oxidation involves the oxidation of lipids, while in in vivo systems free radicals can also damage proteins, DNA and other molecules (Moure, A., Cruz, J. Franco, D., Dominguez, JM, Sineiro , J. Domínguez, H., Núñez, MJ and Parajó, JJ (2001). Natural antioxidants from residual sources. Food Chemistry, 72: 145-20 171).

As antioxidants, synthetic compounds such as butylhydroxytoluene (BHT) or butylhydroxyanixol (BHA) have been used in the food industry. However, there is a growing interest in replacing these synthetic antioxidants with natural antioxidants that have identical mechanisms of action. The worldwide demand for natural antioxidants has increased due to doubts about the safety of food and synthetic food additives and the public's feeling that food and natural food additives provide certain health benefits. Most of the natural compounds with antioxidant activity are found in plants, and many of them are polyphenolic in nature. The term polyphenols represents a collective name for aromatic compounds that contain at least two hydroxyphenolic groups in the molecule (Römpp Lexikon Chemie, 10th edition, 1999, editorial Georg Thieme, Stuttgart, page 3491). This group of compounds includes flavonoids, a heterogeneous group of plant polyphenols (more than 4000) that share a benzopyran structure. They constitute pigments (orange, red and blue colors) of various vegetables (flowers, fruits, leaves) and are important factors for the growth, development and protection of plants. 5 The main flavonoids with antioxidant activity present in vegetables are: (+) - catechin, (-) - epicatechin, (-) - epigallocatechin and the corresponding esters of gallic acid: gallate of (-) - epicatechin and gallate of (-) -epigalocatequina [Blot et al. Am. J. Cancer Prevention 62: 1477-82, 1992].

The raw materials of residual origin of agro-industrial processes that can serve as sources of antioxidants deserve particular attention. In this field, some materials have been considered as raw materials such as residues of potato skin, olive pomace, alpechines, grape seeds and wine bagasse and grape skins. Studies have been conducted that include the identification of active polyphenolic compounds in 15 apple bagasse (Lu, Y. & Foo, LY (1997). Identification and quantitation of major polyphenols in apple pomace. Food Chemistry, 59, 187-194), citrus seeds and skins (Bocco, A., Cuvelier, ME, Richard, H., & Berset, C. (1998). Antioxidant activity and phenolic composition of citrus peel and seed extracts. Journal of Agricultural and Food Chemistry, 46, 2123-2129), coconut by-products (Azizah, AH, Nik Ruslawati, NM, & 20 Swee Tee, T. (1999). Extraction and characterization of antioxidant from cocoa by-products. Food Chemistry, 64, 199-202).

The acorn is the characteristic fruit of the Quercus genus species. Within this genus there are numerous species that give acorns, such as holm oak, oak or cork oak. This type of tree is common in the west of the Iberian peninsula where the largest area of pasture is concentrated. Acorn maturation occurs in autumn-winter providing an important food resource to wildlife and domestic wildlife. Special attention requires the use of acorns as feed for pigs, a species that has been able to obtain greater use of the nutritional values of the acorn when peeled before consumption. This peculiarity has caused that the acorn is preferably reserved for the Iberian pig and that its young in the pasture is centered in its fattening with acorn (montanera), fundamentally of oak (Quercus ilex rotundifolia). The use of acorn in the formulation of feed as well as its importance in animal fattening, has placed this fruit among the most appreciated in the animal feed sector. The composition of the acorn varies from one genus to another and throughout its maturation. Around 25% by weight of the fruit corresponds to the peel whose composition contributes a bitter taste that makes this part of the acorn despised making it a by-product that is not used.

With all this acquires a special interest in obtaining an extract that encompasses all these characteristics, that is natural, that is obtained from an unused part of a natural fruit, that has antioxidant activity, that can be applied in food and that preserves to foods that include oxidative deterioration.

Object of the invention

In a first aspect, the present invention relates to a method of obtaining an acorn shell extract (method of the invention), which comprises the following steps:

a) Crush the acorn shell, 20

b) Add to the crushing of the acorn shell obtained in step a) a solvent selected from the group consisting of water, C1-C6 alcohol and mixtures thereof,

c) Stir the mixture of the crushed and the solvent obtaining two phases, and

d) Separate the two phases obtained in step c) and collect the supernatant comprising the extract. 25

Likewise, in a second aspect, the present invention refers to an acorn shell extract obtainable by the process according to the first aspect of the invention.

A third aspect of the invention relates to the use of the acorn shell extract according to the second aspect of the invention as an antioxidant.

Finally, a fourth aspect of the invention relates to a food, cosmetic or pharmaceutical product comprising the acorn shell extract according to the second aspect of the invention.

Description of the invention 5

Based on the needs of the state of the art, the inventors of the present invention have developed a process for obtaining an acorn shell extract with a high polyphenol content which comprises an extraction stage with a solvent. The extract obtained with the process of the invention has a high activity as an antioxidant.

Thus, in a first aspect, the present invention relates to a method of obtaining an acorn shell extract, comprising the following steps:

a) Crush the acorn shell, 15

b) Add to the shell crushing obtained in step a) a solvent selected from the group consisting of water (acidified or not), C1-C6 alcohol and mixtures thereof,

c) Stir the mixture of the crushed and the solvent obtaining two phases,

d) Separate the two phases obtained in step c) and collect the supernatant comprising the extract.

Thus, the first stage of the process of the invention consists in crushing the acorn shell. The acorn shell is separated by manual or mechanical means. In step a), the crushing of the shell is carried out by manual or mechanical means until small particles are obtained, preferably between 0.1 and 10 mm.

In step b), the proportions used in the shell or solvent can be modified in order to obtain extracts with a greater or lesser concentration and therefore with different antioxidant activity. Preferably, the ratio between the crushed shell and the solvent (shell: solvent) is between 1:50 (weight: volume) and 1: 2 (weight: volume). In a particular embodiment, the shell: solvent ratio is 1:10 (g: mL).

The use of one or another solvent influences the amount of compounds extracted as well as the type of compound with antioxidant activity that is obtained due to their characteristics and their affinity for the solvent. In a preferred embodiment, in step b) the selected solvent is a mixture of water and a C1-C6 alcohol.

In the context of the present invention, the term "C1-C6 alcohol" refers to an alcohol that can have from one carbon to six carbons in a straight or branched chain, with at least one hydroxide function. 10

In a particular embodiment, in step b), the solvent selected is a mixture of distilled water and ethanol, where the water: ethanol ratio is between 20:80 and 80:20 (v: v), more preferably between 30:70 and 70:30 (v: v) and more preferably still 50:50 (v: v). The extraction carried out with a mixture of water and ethanol in a 50:50 ratio is that which results in an extract with a higher content of phenolic and flavonoid compounds.

The term "phenolic compounds" refers to aromatic compounds comprising at least two hydroxyphenolic groups in the molecule. This group of compounds includes flavonoids, a heterogeneous group of plant polyphenols that share a benzopyran structure. The main flavonoids with antioxidant activity are (+) - catechin, (-) - epicatechin, (-) - epigallocatechin and the corresponding esters of gallic acid, (-) - epicatechin gallate and (-) - epigallocatechin gallate.

The conditions of temperature, time and exposure to light in step c) can be adjusted in a range in which the obtaining of compounds with antioxidant activity is optimal avoiding degradation. Preferably, step c) is carried out at a temperature between 20 ° C and 28 ° C, preferably between 22 ° C and 26 ° C, for a time between 30 and 210 minutes, preferably between 100 and 150 minutes, and under conditions of reduced brightness, preferably darkness. In a particular embodiment, step c) is carried out in dark conditions, at a temperature of 25 ° C and for a time of 2 hours.

By stirring stage c) two phases are obtained from which the supernatant phase that separates from the solid phase is used. In step d), the separation of the two phases formed in step c) to collect the supernatant comprising the extract is carried out by decantation or centrifugation or any other method that allows the differentiation of the phases.

The supernatant collected in step d), which comprises the acorn shell extract, may comprise some rest of the crushing or impurities, so that in a particular embodiment, the process of the invention comprises a step e) of filtering the supernatant collected in stage d). Also, the supernatant collected in stage d) or filtered in stage e) can be concentrated by distillation or transformed by lyophilization or any other modification that allows obtaining an extract that suits the application needs. Thus, in another particular embodiment, the process of the invention comprises a stage f) of distillation of the supernatant obtained in step d) or filtered in step e). And in another particular embodiment, the supernatant collected in step d), filtered in step e) or concentrated in step f) is lyophilized (step g). Depending on the final extract, its conservation may be carried out under ambient conditions or it will require controlled temperature storage in an appropriate container. In a particular embodiment, the extract is preserved under dark and cooling conditions.

A second aspect of the invention relates to an acorn shell extract obtainable by the process of the invention according to any one of the embodiments according to the first aspect of the invention. In a preferred embodiment, the extract has a phenolic compound content between 27.5 and 45.0 mg equivalent of gallic acid per gram of sample. Preferably between 41.6 and 44.7 mg equivalents of gallic acid per gram of sample. In a preferred embodiment the extract of the invention has a content in flavonoid compounds comprised between 6.7 and 15.1 mg equivalent of catechin per gram of sample. Preferably between 12.32 and 14.3 mg equivalent of 30 catechin per gram of sample.

The extract of the present invention, obtainable by the process according to any one of the embodiments of the first aspect of the invention, is effective as an antioxidant agent, with important applications in the food, cosmetic and pharmaceutical industry. Thus, a third aspect of the invention relates to the use of the acorn shell extract according to the second aspect of the invention as an antioxidant. In addition, a fourth aspect of the invention refers to a food, cosmetic or pharmaceutical product comprising the extract according to the second aspect of the invention.

The incorporation of the extract into a product is carried out in multiple ways, such as by addition within a formulation, by spraying on the product or any other method that allows the correct distribution of the extract and its antioxidant action. The concentration of extract to be used is determined by factors specific to the extract, such as antioxidant capacity, as well as those attributable to the product such as type of product, content and type of fat, concentration in anti-and pro-oxidants, etc. Likewise, the technological processes that can be carried out after the addition of the extract as well as the product conservation characteristics must be taken into account. Depending on the final product, its conservation may be carried out in environmental conditions or it will require controlled temperature storage in an appropriate container.

Examples 20

The following is an example of obtaining four extracts from acorn husks and their characterization by means of total phenolic content and their flavonoid content in two maturation stages of the acorn (green and ripe).

Example 1. Obtaining the acorn shell extract by using solvent mixture.

To obtain the extract, the acorn shell (Gen. Quercus) was obtained from the pasture itself in two stages of maturation of the acorn, green and ripe. The shells 30 were obtained with the help of a knife separating them from the pulp of the acorn. Later they crumbled by means of chopped in conventional chopper. The extraction of compounds with antioxidant properties was carried out by using two solvents and mixing both. Thus, distilled water, ethanol and two mixtures of water were used: ethanol in a 50:50 and 70:30 (vol: vol) ratio, which gave rise to four different extracts for the different stages of maturation. A 1:10 ratio (g: mL) of shell and solvent used, presented the best results in previous experiences, so it was 5 that was used. The solvent and shell mixtures were kept under stirring for two hours at a controlled temperature of 25 ° C under reduced light conditions. Once the stirring time elapsed, the phases were separated by centrifugation, obtaining two perfectly differentiated phases. The supernatant phrase was filtered through Whatman No. 54 paper filter and stored in an opaque container at refrigeration temperature.

Example 2. Characterization of four acorn shell extracts in two stages of maturation by quantifying their total phenolic content.

The quantification of total phenols of acorn shell extracts was carried out following the procedure described by Singleton et al. (1999). 50uL of each of the extracts were deposited in a microtiter plate well to which 20uL of Folin-Cicalteau reagent and 50uL of 20% sodium carbonate (w / v) were added. The mixture was incubated for one hour at room temperature, after which the absorbance at 765 nm was measured against a blank where the sample was replaced by distilled water. The quantification of the measurements carried out was performed against a standard curve of gallic acid. The results were expressed as equivalents of gallic acid and are reflected in Table 1.

Example 3. Characterization of four acorn shell extracts in two stages of maturation by quantifying their flavonoid content.

The quantification of the flavonoid content of the extracts obtained from the acorn shell was carried out following the method proposed by Zhishen et al. (1999). This procedure is based on the formation of chelates between AlCl3 and flavonoids in the sample, which have a pinkish color. Thus, 400uL of sample was deposited in a test tube to which 60uL of 10% NaNO2, 60uL of 20% AlCl3 and 400uL of 1M NaOH were added. After stirring its absorbance was measured at 510 nm. The quantification of the measurements performed was performed against a catechin standard curve. The results were expressed in mg of equivalent catechin per gram of sample and are reflected in Table 1.

Table 1. Phenolic compound content (mg Eq gallic acid / g sample) and flavonoid content (mg Eq. Catechin / g sample) of extracts (water, ethanol, water mixture: 50:50 v / v ethanol and water mixture : ethanol 70:30 v / v) of acorn shell.

 SEM Extraction Method Sig.

 Water Ethanol 50:50 70:30

 Phenolic compounds

 41.60 b 28.21 d 44.71 to 39.20 c 0.33 ***

 Flavonoids

 12.32 ab 7.46 c 14.31 a 11.88 b 0.75 ***

N = 5 determinations

Sig .: Level of significance ANOVA test; ***: p <0.001. a, b, c: In the same row, means with different letters imply statistically significant differences (Tukey test, p <0.05)

The evaluation of the antioxidant capacity of acorn shell extracts was carried out following two synthetic radical reduction methodologies, FRAP method and ABTS method, as shown in examples 4 and 5. These tests demonstrate the antioxidant power of the sample by changes in the coloration of a solution containing a radical that can be reduced. In both cases, the response obtained is extrapolated to a Trolox calibration curve, so the results are expressed in 20 TEAC (antioxidant activity equivalent to Trolox).

Example 4. Evaluation of the antioxidant capacity of four acorn shell extracts by using the ABTS radical.

The method proposed by Re et al. (1999). Thus, the ABTS radical was obtained from the equal part mixing of a solution of ABTS / nM and 2.45nM potassium persulfate. This solution was kept at room temperature for 16 hours in dark conditions for the generation of the radial. The solution was diluted with ethanol to obtain an absorbance of 0.7 (± 0.1) at 734 nm. 245uL of the solution with the ABTS radical was deposited in a well of a microtiter plate and its absorbency was measured at 734nm. Then 5uL of the extract to be tested was added and, after one minute, the measurement was repeated at 734 nm. The results obtained faced a calibration curve of 5 Trolox. The results were expressed as mg TEAC / g sample and are reflected in table 2.

Example 5. Evaluation of the antioxidant capacity of four acorn shell extracts using the FRAP method. 10

This method proposed by Benzie and Strain (1996), determines the capacity of ferric reduction that a sample has at low pH, transforming the tripyridyltriazine complex (TPTZ) with iron (III) to its ferrous form. Thus, the FRAP reagent was prepared from 2.5 ml of a 10mM TPTZ solution together with 2.5mL of a 20mM FeCl3-6H2O solution and 25 mL 15 of 0.3mM acetate buffer and pH 3.6. A 593nm absorbance reading of 200mL of the FRAP reagent was made to which 7uL of the extract to be tested was subsequently added, taking absorbency measurements after 30 minutes. The results obtained faced a Trolox calibration curve. The results were expressed as mg TEAC / g sample and are reflected in table 2. 20

Table 2. Antioxidant capacity measured by the ABTS method (mg TEAC / g of sample) of extracts (water, ethanol, water mixture: 50:50 v / v ethanol, and water mixture: 70:30 v / v ethanol) of acorn shell.

 SEM Extraction Method Sig.

 Water Ethanol 50:50 70:30

 ABTS

 58.14 b 42.33 c 80.22 a 73.34 a 2.71 ***

 FRAP

 146.76 to 115.66 b 159.71 to 157.1 to 7.89 ***

N = 5 determinations

Sig .: Level of significance ANOVA test; ***: p <0.001. a, b, c: In the same row, means with different letters imply statistically significant differences (Tukey test, p <0.05)

Claims (16)

1. Procedure for obtaining an acorn shell extract comprising the following stages: to. Crush the acorn shell, 5 b. Add to the crushing of the acorn shell obtained in step a) a solvent selected from the group consisting of water (acidified or not), C1-C6 alcohol and mixtures thereof, C. Stir the mixture of the crushed and the solvent obtaining two phases, and d. Separate the two phases obtained in step c) and collect the supernatant comprising the extract. 2. A method according to claim 1, comprising a step e) of filtration of the supernatant collected in step d). 3. The method according to claim 1 or 2, comprising a stage f) of distillation of the supernatant collected in step d) or of the filtrate obtained in step e). fifteen 4. Method according to any one of claims 1 to 3, comprising a step g) of lyophilization of the supernatant collected in step d) or of the filtrate obtained in step e) or of the distillate obtained in step f). 5. Method according to any of claims 1-4, wherein, in step a), the shell is crushed to a particle size between 0.1 and 10 mm. twenty 6. Method according to any one of claims 1 to 5, wherein the crushed shell-solvent ratio is between 1:50 (weight: volume) and 1: 2 (weight: volume). 7. Process according to any one of claims 1 to 6, wherein the solvent selected is a mixture of water and a C1-C6 alcohol. 25 8. Method according to claim 7, wherein the water: C1-C6 alcohol ratio is between 20:80 and 80:20 (v / v). 9. Process according to claim 7 or 8 wherein the solvent selected in step b) is a mixture of water and ethanol. 10. Method according to any one of claims 1-9, wherein step c) 30 is carried out at between 20 and 28 °, for 30 and 210 minutes. 11. Acorn shell extract obtainable by the process according to any one of claims 1-10. 12. Acorn shell extract according to claim 11, characterized in that it has a total phenolic content between 27.5 and 45.0 mg equivalent of gallic acid / g of sample. 13. Acorn shell extract according to claim 11 or 12 characterized in that 5 has a flavonoid content comprised between 6.7 and 15.1 mg equivalents of gallic acid / g of sample. 14. Use of the acorn shell extract according to any of claims 11-13 as an antioxidant. 15. Food, cosmetic or pharmaceutical product comprising the acorn shell extract of claims 11-13.